In each division cycle of a normal somatic animal cell, a crucial block to further proliferation has to be passed at the point where the decision is made to replicate the genetic material, DNA. In contrasts, in cancer cells, proliferation control is deranged. It is therefore of great importance to identify the molecules which trigger this decision point and to understand the signals upon which their action depends. Drosophila is an excellent organism in which to investigate these molecular checkpoints using genetics, cell biology and molecular probes. The phenotypes of mutations at the pan gu (png) locus of Drosophila indicate that this gene is of key importance in te regulation of embryonic DNA replication. png is one of three, interacting, maternal genes whose products are essential to prevent DNA replication in the unfertilized egg. In fertilized eggs from png mutant mothers DNA synthesis is uncoupled from mitosis, producing giant, polyploid nuclei. The aim of this proposal is to determine the molecular mechanism whereby DNA synthesis is repressed, and how this repression is overcome by fertilization. To do this it will be necessary to clone the png gene and to analyze its sequence and spatial and temporal expression. The role of the png+ product in coupling DNA synthesis to mitosis will be tested by determining whether DNA synthesis in png mutant embryos occurs in discrete cycles. For this, nucleotide pulse-labelling will be used in vivo and in extracts. The dependence of one gene product upon another will be inferred from the effect of duplications and mutations of other genes upon both the png mutant phenotype and the distribution of the png+ product. These experiments will locate the wild type png+ gene product within the hierarchy of genes coordinating DNA replication with meiosis, fertilization and mitosis in the early embryo.